Identification of PP2A as a novel interactor and regulator of TRIP-Br1.
TRIP-Br proteins are a novel family of transcriptional coregulators involved in E2F-mediated cell cycle progression. Three of the four mammalian members of TRIP-Br family, including TRIP-Br1, are known oncogenes. We now report the identification of the Balpha regulatory subunit of serine/threonine protein phosphatase 2A (PP2A) as a novel TRIP-Br1 interactor, based on an affinity binding assay coupled with mass spectrometry. A GST-TRIP-Br1 fusion protein associates with catalytically active PP2A-ABalphaC holoenzyme in vitro. Coimmunoprecipitation confirms this association in vivo. Immunofluorescence staining with a monoclonal antibody against TRIP-Br1 reveals that endogenous TRIP-Br1 and PP2A-Balpha colocalize mainly in the cytoplasm. Consistently, immunoprecipitation followed by immunodetection with anti-phosphoserine antibody suggest that TRIP-Br1 exists in a serine-phosphorylated form. Inhibition of PP2A activity by okadaic acid or transcriptional silencing of the PP2A catalytic subunit by small interfering RNA results in downregulation of total TRIP-Br1 protein levels but upregulation of serine-phosphorylated TRIP-Br1. Overexpression of PP2A catalytic subunit increases TRIP-Br1 protein levels and TRIP-Br1 co-activated E2F1/DP1 transcription. Our data support a model in which association between PP2A-ABalphaC holoenzyme and TRIP-Br1 in vivo in mammalian cells represents a novel mechanism for regulating the level of TRIP-Br1 protooncoprotein.
Zang
Zhi Jiang
ZJ
Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Gunaratnam
Lakshman
L
Cheong
Jit Kong
JK
Lai
Li Yun
LY
Hsiao
Li-Li
LL
O'Leary
Eileen
E
Sun
Xiaoming
X
Salto-Tellez
Manuel
M
Bonventre
Joseph V
JV
Hsu
Stephen I-Hong
SI
D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D047468:Immunoprecipitation; D008163:Luminescent Measurements; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D000092468:Bromodomain Containing Proteins
D009687:Nuclear Proteins; D019319:Okadaic Acid; D054648:Protein Phosphatase 2; D034741:RNA, Small Interfering; D015534:Trans-Activators; D014157:Transcription Factors; C485730:Sertad1 protein, mouse; D000092468:Bromodomain Containing Proteins